Collage-based therapeutic delivery systems

ABSTRACT

A collagen-based therapeutic delivery device includes an insoluble synthetic collagen-fibril matrix comprising a polymerization product of soluble oligomeric collagen or a polymerization product of a mixture of soluble oligomeric collagen with one or more type of non-oligomeric soluble collagen molecules, such as, for example, soluble telocollagen and/or soluble atelocollagen, and an active agent dispersed throughout the collagen-fibril matrix or within a portion of the collagen-fibril matrix. A pre-mat rix composition includes an aqueous solution including soluble collagen-fibril building blocks and an active agent in the aqueous solution. The soluble collagen-fibril building blocks include soluble oligomeric collagen or a mixture of soluble oligomeric collagen with non-oligomeric soluble collagen molecules. The building blocks are operable to self-assemble into a macromolecular synthetic collagen-fibril matrix in the absence of an exogenous cross-linking agent. Methods of making and using the pre-matrix composition and the device are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/042,664, filed Aug. 27, 2014, entitled “Drug DeliverySystem,” the disclosure of which is expressly incorporated by referenceherein in its entirety.

TECHNICAL FIELD

The present disclosure generally relates to a therapeutic deliverydevice comprising a synthetic collagen-fibril matrix with an activeagent dispersed therein, a pre-matrix solution operable for making adelivery device and methods for active agent delivery. The disclosurealso relates to methods of customizing a delivery system by controllingone or more of the proteolytic biodegradability properties of thecollagen-fibril matrix, the microstructural properties of thecollagen-fibril matrix, the mechanical properties of the collagen-fibrilmatrix and the transport properties of the collagen-fibril matrix.

BACKGROUND OF THE DISCLOSURE

Collagen, the major extracellular matrix (ECM) component of connectivetissues, has received a great deal of attention as a candidate materialfor use as an implantable or injectable drug delivery vehicle, primarilybecause of its biocompatibility, low immunogenicity andbiodegradability. Extracellular matrices are known to providescaffolding for cells, while organizing the cells three-dimensionallyand providing essential information to regulate cell behavior. As such,the field of tissue engineering strives to mimic both the form andfunction of these scaffolds to create compositions for optimal tissuerepair and replacement. Collagen, and in particular type I collagen, maybe used in the field of tissue engineering due to its high availabilityin the body, conservation across tissues and species, biodegradabilityand biocompatibility. In fact, not only is collagen the most abundantmolecule of the ECM∞ it is responsible for the majority of thestructural and mechanical properties of several tissues. The in vivoform of collagen is a triple-helix center region that is capped at bothends by randomly organized telopeptides. These collagen molecules arefound within the ECM assembled as branched collagen-fibril networks thatcontain natural molecular cross-links.

In spite of numerous advantages and wide research on collagen as anatural biomaterial, its use as a vehicle for controlling local activeagent release has been limited. Furthermore, its application as a tissuegraft that induces appropriate tissue regeneration while at the sametime achieving predictable localized delivery of specified agents hasnot been achieved to date. In fact, only a few collagen-based activeagent delivery formulations have made it into clinical trials. Existingformulations can be categorized as either non-dissociated fibrillarcollagens or solubilized collagens. These formulations have a number ofshortcomings, including poorly defined molecular composition, lowmechanical integrity, rapid biodegradation and limited control over drugrelease profiles.

Non-dissociated fibrillar collagens are formulations that containdecellularized collagen extracellular matrix (ECM) particulate matter,which is mechanically homogenized, acid-swollen, and finally lyophilizedto form sponge that may or may not be cross-linked. Soluble collagens,by contrast, are obtained from pepsin or acid solubilization ofmammalian tissues to form viscous collagen solutions, which are thenlyophilized and formulated as a cross-linked or non-cross-linked spongeor injectable viscous gel. As stated above, previously-describedcollagen-based active agent delivery platforms have many limitations,including poorly defined molecular compositions, low mechanicalintegrity and stability, rapid proteolytic degradation rapid proteolyticdegradation and limited design control. While exogenous crosslinking,including chemical and physical means, is routinely used to improvemechanical and handling properties as well as increase persistence uponimplantation, such processing is associated with deleterious tissueresponses and loss of biological activity.

The marginal success of these present day collagen-based drug deliveryformulations can be traced to these major limitations. Moreover, theseconventional formulations exhibit amorphous microstructures, withunsatisfactory control of material properties, including pore size andproteolytic degradability. Cursory control of these parameters is oftenachieved through modulation of lyophilization conditions and/orexogenous chemical and physical crosslinking. Materials formed withoutcross-linking represent viscous gels. They are characterized asmechanically unstable, too soft to handle, and unable to resistcell-induced contractions, thus failing to support cell ingrowth andmigration required for tissue regeneration. On the other hand, exogenouscrosslinking has been shown to have detrimental effects on cells andtissues, such as cytotoxicity or tissue calcification and partialdenaturation of collagen itself. Aldehyde based cross-linking may leadto aldehyde residues in the final product and may influence thebiocompatibility of the collagen. Moreover, de-hydrothermalcross-linking has natural limitations and does not lead to materialswith sufficiently improved properties.

Accordingly, there is a need for further advancements in the design anddevelopment of local therapeutic delivery systems and integrated tissueregeneration. As will be explained in detail herein, the presentdisclosure addresses this need and also provides associatedcompositions, devices and methods that address deficiencies in theexisting art.

SUMMARY OF THE DISCLOSURE

In accordance with one aspect of the present disclosure, a therapeuticdelivery device is provided that includes an insoluble syntheticcollagen-fibril matrix and an active agent dispersed throughout thecollagen-fibril matrix or within a portion of the collagen-fibrilmatrix. The collagen-fibril matrix comprises a polymerization product ofsoluble oligomeric collagen or a polymerization product of a mixture ofsoluble oligomeric collagen with one or more other type of solublecollagen molecules, also referred to herein as non-oligomeric solublecollagen molecules. In one embodiment, the non-oligomeric solublecollagen molecules include one or more of soluble telocollagen moleculesand soluble atelocollagen molecules. In one embodiment, the syntheticcollagen-fibril matrix exhibits a stiffness of at least 5 Pa. Theoligomeric collagen or mixture of soluble oligomeric collagen with oneor more type of non-oligomeric soluble collagen molecules is capable ofself-assembling into a synthetic macromolecular collagen-fibril matrixin the absence of an exogenous crosslinking agent.

In another aspect of the disclosure, a method for making a therapeuticdelivery device is provided that includes (i) forming an aqueoussolution comprising a quantity of soluble collagen-fibril buildingblocks; (ii) causing the building blocks to polymerize by self-assembly,thereby forming an insoluble synthetic collagen-fibril matrix; and (iii)either (a) including a quantity of an active agent in the aqueoussolution whereby said causing forms a collagen-fibril matrix having theactive agent dispersed therein or (b) contacting the syntheticcollagen-fibril matrix with the quantity of the active agent to form acollagen-fibril matrix having the active agent dispersed therein. Thequantity of building blocks comprises soluble oligomeric collagen. Inone embodiment, the collagen-fibril matrix exhibits a stiffness of atleast 5 Pa.

This disclosure also provides a pre-matrix composition that comprises anaqueous solution that includes soluble collagen-fibril building blocksand an active agent. The soluble collagen-fibril building blocks includesoluble oligomeric collagen or a mixture of soluble oligomeric collagenwith one or more type of non-oligomeric soluble collagen molecules,which are operable to self-assemble into a synthetic macromolecularcollagen-fibril matrix in the absence of an exogenous cross-linkingagent. In one embodiment, the non-oligomeric soluble collagen moleculesinclude one or more of soluble telocollagen molecules and solubleatelocollagen molecules. In one embodiment, the agent is associated withone or more collagen-fibril building blocks. In one embodiment, thecollagen-fibril matrix exhibits a stiffness of at least 5 Pa.

In another aspect of the disclosure, there is provided a method fordelivering an active agent that includes positioning at an in situlocation (i) a pre-matrix composition that comprises an aqueous solutionthat includes soluble collagen-fibril building blocks and an activeagent; or (ii) a therapeutic delivery device that includes an insolublesynthetic collagen-fibril matrix and a first active agent dispersedthroughout the collagen-fibril matrix or within a portion of thecollagen-fibril matrix.

Still other embodiments and features of the application will becomeapparent from the following written description along with theaccompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

The various aspects of the present application will become more apparentand the teachings of the present application itself will be betterunderstood by reference to the following description of the embodimentsof the present application taken in conjunction with the accompanyingdrawings, wherein:

FIG. 1 depicts a schematic of example soluble building blocks of acollagen-fibril matrix in accordance with the teachings of the presentdisclosure;

FIG. 2 depicts an illustrative representation of the formation acollagen matrix-based delivery system in a well of a 48 well plate asdescribed in Example 1 of the present disclosure, and wherein thedelivery device, in turn, is placed in a reservoir of phosphate bufferedsaline (PBS) in the presence or absence of collagenase to measureexperimentally its agent release profile;

FIG. 3 depicts a graphical representation of the effect of admixedFITC-Dextrans (2 mg/ml) on the collagen-fibril polymerization kinetics(A) and physical properties (B) of the collagen-fibril matrix deliverydevice as described in Example 2 of the present disclosure;

FIG. 4 depicts an illustrative representation of size-dependentmolecular release kinetics as predicted using a diffusion-basedmathematical model as described in Example 3;

FIG. 5 represents data obtained from analyzing the molecular release ofpolymerizable oligomer collagen and commercial monomer collagen (BD rattail) matrices as described in Example 4 of the present disclosure;

FIG. 6 represents data obtained from analyzing the molecular release ofoligomer collagen-fibril compositions and commercial monomercollagen-fibril matrices in the presence of collagenase as described inExample 4 of the present disclosure;

FIG. 7 represents data obtained from an oscillatory shear-based, andstrain-controlled time sweep experiment with polymerized atelocollagen(squares), telocollagen (circles) or oligomer (triangles) matricesexposed to 5000 U/ml collagenase as described in Example 4 of thepresent disclosure;

FIG. 8 represents data obtained from analyzing the molecular release ofoligomer, telocollagen and atelocollagen matrices (3 mg/ml) polymerizedwith 10 kDa or 2 MDa FITC-dextran molecules as described in Example 4 ofthe present disclosure;

FIG. 9 depicts polymerization profile data obtained during thepolymerization of collagen-fibril matrices as described in Example 6 ofthe present disclosure;

FIG. 10 represents time release profiles of small (10 kDa; panels A andB) and large (2 MDa; panels C and D) FITC-Dextran molecules polymerizedwithin a variety of collagen-fibril matrices as described in Examples 7and 10 of the present disclosure;

FIG. 11 depicts a summary of data obtained from FITC-dextran releasefrom low-density (3 mg/ml) and high-density (15.6 mg/ml) collagen-fibrilmatrices in the presence of 50 U/ml collagenase as described in Example10 of the present disclosure;

FIG. 12 depicts a schematic comparing the size of fluorescein andindicated FITC-dextran molecular weights relative to a variety ofpotential active agents in accordance with the teachings of presentdisclosure;

FIG. 13 depicts a previously published chart comparing FITC-Dextranparticle size (MW) with hydrodynamic radius (nm);

FIG. 14 depicts a schematic of a method of creating high densitycollagen-fibril matrices using confined compression as described inExample 9 of the present disclosure;

FIG. 15 represents release kinetics data obtained from low-density (3mg/ml) oligomer matrices prepared with either 10 kDa (left panel) or 2MDa (right panel) FITC-Dextran and treated with the indicatedcollagenase concentration as described in Example 4 of the presentdisclosure;

FIG. 16 depicts illustrative graphs showing the initial rates of releaseand T50% curves calculated from the release kinetics curves of FIG. 15for the various collagenase concentrations;

FIG. 17 depicts illustrative graphs showing a summary of data obtainedfor 10 kDa FITC-dextran release from oligomer collagen-fibril matricesprepared over a broad range of densities (3 to 40 mg/ml) in the absenceof collagenase as described in Example 11 of the present disclosure;

FIG. 18 depicts illustrative graphs showing a summary of data obtainedfor 10 kDa FITC-dextran release from oligomer collagen-fibril matricesprepared over a broad range of densities (3 to 40 mg/ml) in the presenceof collagenase (10 U/ml) as described in Example 11 of the presentdisclosure; and

FIG. 19 depicts illustrative graphs showing a summary of data obtainedfor 2 MDa FITC-dextran release from oligomer collagen-fibril matricesprepared over a broad range of densities (3 to 40 mg/ml) in the presenceof collagenase (10 U/ml) as described in Example 11 of the presentdisclosure.

Although the exemplification set out herein illustrates embodiments ofthe present application in several forms, the embodiments disclosedbelow are not intended to be exhaustive or to be construed as limitingthe scope of the present application to the precise forms disclosed.

DETAILED DESCRIPTION

The embodiments of the present disclosure that are described herein arenot intended to be exhaustive or to limit the teachings of the presentapplication to the precise forms disclosed in the following detaileddescription. Rather, the embodiments are chosen and described so thatothers skilled in the art may appreciate and understand the principlesand practices of the present disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present application, various specificmethods and materials are now described.

One aspect of the present disclosure is directed to a collagen-basedtherapeutic delivery device comprising an insoluble syntheticcollagen-fibril matrix with an active agent dispersed therein. Inanother aspect, the disclosure is directed to a pre-matrix composition.In still other aspects, the disclosure provides methods of making andusing the therapeutic delivery device and the pre-matrix composition. Inaccordance with certain embodiments of the present disclosure, anillustrative therapeutic delivery device is prepared by polymerizingdefined mixtures of collagen-fibril matrix building blocks from aqueoussolution. The collagen based therapeutic delivery device can beimplantable or injectable. The disclosed systems enable a broad range ofcustomizable spatiotemporal molecular release profiles for one or moretherapeutics, referred to herein as active agents, including burst,sustained and targeted release.

As used herein, the term “collagen-fibril matrix” or “collagen-fibrilmaterial” refers to a Type I collagen composition that includes collagenfibrils and that has been formed under controlled conditions fromsolubilized collagen building blocks. In one embodiment, at least 1% ofthe collagen in the collagen-fibril matrix is composed of oligomers. Inother embodiments, at least 2%, at least 3%, at least 4% or at least 5%of the collagen in the collagen-fibril matrix is composed of oligomers.In one embodiment, the collagen-fibril matrix material has a stiffnessof at least 5 Pa. In other embodiments the collagen-fibril matrixmaterial has a stiffness of at least 10 Pa, at least 15 Pa, at least 20Pa or at least 25 Pa.

As used herein, the term “collagen” refers to a family of at least 20genetically different secreted proteins that serve a predominantlystructural function and possess a unique triple helical structureconfiguration of three polypeptide units known as alpha chains. Thethree alpha chains (two α1 (I) and one α2(I) chain) are characterized by(Gly-X-Y)_(n) repeat units where the X and Y positions are oftenoccupied by proline and hydroxyproline forming an approximately 300 nmlong triple helical collagen molecule flanked on each end by anon-helical telopeptide end. These collagen molecules, also known asmonomers, are the fundamental building blocks that self-assemble in ahierarchical fashion to form tissue-specific networks of micro-fibrils,fibrils, fiber and fiber bundles that then combine to form the ECM ofthe body's tissues. Lysyl oxidase binds to and catalyzes cross-linkformation between prefibrillar aggregates of staggered collagenmolecules (called monomers) to create covalently cross-linked dimers ortrimers (called oligomers). The different oligomer precursors modulatethe progressive molecular packing and assembly that eventually yieldstissue-specific fibril architecture and matrix function. Both monomerand oligomer formulations possess intact telopeptide regions and containreactive aldehydes generated from acid-labile, intermediate cross-links.Proteolytic enzyme treatment of collagen removes the terminaltelopeptide regions to yield atelocollagen formulations, whereas removalof the amino and carboxyl-telopeptides results in an amorphousarrangement of collagen molecules and loss of the banded-fibril patternin a reconstituted product.

As used herein, the term “oligomer” refers to a molecule in which two ormore tropocollagen molecules are covalently attached to one another viaa naturally occurring intramolecular cross-link and which is soluble inan aqueous fluid.

As used herein, the term “telocollagen” (also referred to as“tropocollagen”, “telomer”, a “collagen monomer” or “monomericcollagen”) refers to an individual collagen molecule in which carboxy-and amino-terminal non-helical telopeptide ends are intact; which isable to undergo self-assembly into a fibrillar matrix, and which lacksintermolecular covalent cross-links.

As used herein, the term “telopeptide” refers to amino- andcarboxy-terminal non-triple helical domains of tropocollagen strandsknown to be important to fibrillogenesis, polymerization andlysyl-oxidase mediated intermolecular cross-link formation.

As used herein, the term “atelocollagen” refers to a triple helixmolecule in which the telopeptide regions have been partially orcompletely removed from tropocollagen. Such atelocollagen preparationsare typically the outcome of enzyme-based (for example, pepsin-based)collagen extraction procedures from tissues.

As used herein, the term “collagen mimetic peptides” refers tochemically-synthesized collagen building blocks having specific aminoacid sequences representing the triple helical portion of collagen,often -(Pro-Hyp-Gly)-, forms a triple helix conformation that resemblesthat found in natural collagens.

As used herein, the term “matrix” refers to a loose meshwork withinwhich cells are or can be embedded or an arrangement of connectedthings. In the context of collagen, matrix refers to a compositematerial composed of an insoluble collagen-fibril network or amorphousnanostructure surrounded by an interstitial fluid phase.

It should be understood and appreciated herein that oligomers comprisesmall aggregates of collagen molecules (e.g., dimers or trimers), whichretain collagen's tissue-specific, covalent intermolecular cross-links,whereas telocollagen (or monomers) are individual collagen moleculesthat lack these intermolecular covalent cross-links. Telocollagen andoligomers possess intact telopeptide regions and contain reactivealdehydes generated from acid-labile, intermediate cross-links. Uponnatural in vivo polymerization, the process through which collagenfibrils assemble to form a fibril polymeric network, these reactivealdehydes spontaneously reform covalent, intermediate cross-links aspart of the fibril-forming process. Pepsin-solubilized(telopeptide-deficient) atelomer (or atelocollagen) formulations arecreated when collagen is treated with proteolytic enzymes that removethe terminal telopeptide regions. As both the amino (N)- and carboxyl(C)-telopeptides play important roles in cross-linking and fibrilformation, their complete removal results in an amorphous arrangement ofcollagen molecules and a consequent loss of the banded-fibril pattern ina reconstituted product.

The collagen matrix building blocks, including oligomers, telocollagenand atelocollagen, may be obtained from a wide variety of raw collagensources known in the art including, but not limited to, mammaliantissues, such as bovine, porcine and equine hides and tendons, and humantendons. Alternatively, the building blocks can be collagen mimeticpeptides or soluble collagen molecules produced using recombinanttechnology. The matrices formed from these building blocks as describedin the present disclosure exhibit superior mechano-biological propertiesas compared to commercially available collagen formulations. Thematrices described herein also exhibit different properties than raw ornative collagen. Type I collagen polymers in oligomer form are a primarybuilding block of the insoluble synthetic collagen-fibril matrixdescribed herein. As further described herein, the ratio of oligomer tonon-oligomeric soluble collagen molecules, such as, for example,atelocollagen and/or telocollagen, can be varied to modulate variousproperties of the formed collagen-fibril matrix, which enablescustomization of a collagen-based therapeutic delivery device asdisclosed herein depending on the intended purpose, location andplacement of the device.

As indicated above, in addition to the collagen-fibril matrix, thecollagen-based therapeutic delivery device also includes an active agentor, optionally, more than one active agent. The one or more active agentincluded in the device also can vary depending on the intended purpose,location and placement of the device. As used herein, the term “activeagent” refers to any compound, agent, molecule, biomolecule, drug,therapeutic agent, nanoparticle, peptide, protein, polypeptide,antibody, ligand, partial antibody, steroid, growth factor,transcription factor, DNA, RNA, virus, bacteria, lipid, vitamin, smallmolecule, or large molecule that has activity in a biological system.Active agents include but are not limited to “biomolecules”, “drugs”,silver amine complexes, surfactants, polyhexamethylene biguanidine,betaine, antimicrobials, linear polymer biguanidines with a germicidalactivity, bioactive additives, heparin, glyosaminoglycans, extracellularmatrix proteins, antibiotics, growth factors, epidermal growth factor(EGF), platelet derived growth factor (PDGF), fibroblast growth factor(FGF), collagen binding peptides or factors, connective tissueactivating peptides (CTAP), transforming growth factors (TGFs),oncostatic agents, immunomodulators, immunomodulating agents,anti-inflammatory agents, osteogeneic agents, hematopoietic agents,hematopoietic modulators, osteoinductive agents, TGF-β1, TGF-β2, TGFα,insulin-like growth factors (IGFs), tumor necrosis factors (TNFs),interleukins, IL-1, IL-2, colony stimulating factors (CSF), G-CSF,GM-CSF, erythropoietin, nerve growth factors (NGF), interferons (IFN),IFN-α, IFN-β, IFN-γ, preservatives, dyes, non-bioactive agents, hormonesand synthetic analogs of the above. “Biomolecules” include, but are notlimited to, steroids, growth factors, transcription factors, DNA, RNA(including siRNA, mRNA etc), peptides (natural and synthetic), proteins,partial or whole antibodies, ligands, viruses, bacteria, lipids, orvitamins. “Drugs” include but are not limited to “small molecules”including but not limited to chemotherapeutics, inhibitors, stimulators,proteases, antibiotics, antivirals; “biomolecules”, “large molecules” ora combination thereof and wherein the drug is used to have a beneficialor negative effect on the target protein, cell or tissue. Surfactantsmay include, but are not limited to, glycine derivatives,sulfosuccinate, and an amide based on an unbranched fatty acid.

As used herein, the term “immunomodulatory amount” refers to an amountof a particular agent or factor sufficient to show a demonstrable effecton the subject's immune system. Immunomodulation may suppress or enhancethe immune system as desired by a practitioner. Suppressing the immunesystem may be desirable when the subject is an organ transplantrecipient or for treatment of autoimmune disease including but notlimited to lupus, autoimmune arthritis, autoimmune diabetes; additionaldiagnoses in which suppression of the immune system is desirable areknown to those skilled in the art. Alternatively, immnunomodulation mayenhance the immune system for example in the treatment of cancer,serious infection or wound repair; additional diagnoses in whichenhancement of the immune system is desirable are known to those skilledin the art.

As used herein, the term “oncostatically effective amount” is an amountof an agent which is capable of inhibiting tumor cell growth in asubject having tumor cells sensitive to the selected agent.

As used herein, the term “hematopoietically modulatory amount” is thatamount of an agent which enhances or inhibits the production and/ormaturation of bloods cells.

As used herein, the term “osteoinductive amount” is that amount of anagent which causes or contributes to a measurable increase in bonegrowth or rate of bone growth.

The collagen-fibril matrix building blocks used to construct thecollagen compositions described herein can be obtained from a number ofsources, including, for example, porcine skin. Suitable tissues usefulas a collagen-containing source material for isolating collagen orcollagen components to make the collagen compositions described hereininclude submucosa tissues or any other extracellular matrix-containingtissues of a warm-blooded vertebrate. Suitable methods of preparingsubmucosa tissues are described in U.S. Pat. Nos. 4,902,508; 5,281,422and 5,275,826, the disclosures of which are each incorporated herein byreference in their entirety. Extracellular matrix material-containingtissues other than submucosa tissue may be used to obtain collagen inaccordance with still other embodiments disclosed herein. Method ofpreparing other extracellular matrix material-derived tissues for use inobtaining purified collagen or partially purified extracellular matrixcomponents are known to those skilled in the art. For example, see U.S.Pat. Nos. 5,163,955 (pericardial tissue); 5,554,389 (urinary bladdersubmucosa tissue); 6,099,567 (stomach submucosa tissue); 6,576,265(extracellular matrix tissues generally); 6,793,939 (liver basementmembrane tissues); and 7,919,121 (liver basement membrane tissues); andInternational PCT Publication No. WO 2001/45765 (extracellular matrixtissues generally), each incorporated herein by reference. In variousother embodiments, the collagen-containing source material can beselected from the group consisting of placental tissue, ovarian tissue,uterine tissue, animal tail tissue, skin tissue, bone, tendon andcartilage tissue. In some embodiments, the collagen is selected from pigskin collagen, bovine collagen and human collagen; however, it should beunderstood and appreciated herein that any suitable extracellularmatrix-containing tissue can be used as a collagen-containing sourcematerial to isolate purified collagen or partially purifiedextracellular matrix components in accordance with the presentteachings.

An illustrative preparation method for preparing submucosa tissues as asource of purified collagen or partially purified extracellular matrixcomponents is described in U.S. Pat. No. 4,902,508, the disclosure ofwhich is incorporated herein by reference. In one embodiment, a segmentof vertebrate intestine, for example, preferably harvested from porcine,ovine or bovine species, but not excluding other species, is subjectedto abrasion using a longitudinal wiping motion to remove cells orcell-removal is accomplished by hypotonic or hypertonic lysis. In oneembodiment, the submucosa tissue is rinsed under hypotonic conditions,such as with water or with saline under hypotonic conditions and isoptionally sterilized. In another illustrative embodiment, suchcompositions can be prepared by mechanically removing the luminalportion of the tunica mucosa and the external muscle layers and/orlysing resident cells with hypotonic or hypertonic washes, such as withwater or saline. In these embodiments, the submucosa tissue can bestored in a hydrated or dehydrated state prior to isolation of thepurified collagen or partially purified extracellular matrix components.In various aspects, the submucosa tissue can comprise any delaminationembodiment, including the tunica submucosa delaminated from both thetunica muscularis and at least the luminal portion of the tunica mucosaof a warm-blooded vertebrate.

As indicated above, one building block used to prepare thecollagen-fibril matrix is oligomeric collagen. The presence ofoligomeric collagen enables the self-assembly of the building blocksinto a collagen-fibril matrix and increases the assembly rate, yieldingcollagen compositions with distinct fibril microstructures and excellentmechanical integrity (e.g., stiffness).

In some embodiments, the building blocks for the collagen-fibril matrixalso include various proportions non-oligomeric soluble collagenmolecules. In one embodiment, the building blocks include one or both oftelocollagen and/or atelocollagen. In certain embodiments, the buildingblocks include oligomeric collagen and atelocollagen. In otherembodiments the building blocks include oligomeric collagen andtelocollagen. In still other embodiments, the building blocks includeoligomeric collagen, telocollagen, and atelocollagen. The amounts ofoligomeric collagen, telocollagen, atelocollagen and/or othernon-oligomeric soluble collagen molecules may be formulated in solutionprior to initiation of polymerization to modulate one or more propertyof the resulting synthetic collagen-fibril matrix including, forexample, stiffness, strength, fluid and mass transport, proteolyticdegradation and/or compatibility. It is recognized that a predeterminedratio of oligomer to non-oligomeric soluble collagen molecules for usewith a particular active agent may differ from that suitable for usewith a different active agent.

Collagen concentration may be expressed in mass/volume or mass/mass.Collagen content may be measured by any means known in the art,including but not limited to, calibrated colorimetric assays such asSirius red and amino acid analysis for hydroxyproline. Viscosity ofcollagen polymer formulations is impacted by a number of factors whichmay include, but are not limited to: solution or dispersion/suspension,concentration, molecular composition, molecular size, temperature andoperating condition. Viscosity measurements may be obtained by any meansknown in the art including, but not limited to, a viscometer orrheometer.

The concentration of soluble collagen present in an aqueous pre-matrixcomposition used to make a synthetic collagen-fibril matrix can vary. Insome embodiments, the collagen is present at a concentration of about0.5 mg/ml to about 500 mg/ml. In other embodiments, the collagen ispresent at a concentration of about 0.5 mg/ml to about 400 mg/ml. In yetother embodiments, the collagen is present at a concentration of about0.5 mg/ml to about 300 mg/ml. In some embodiments, the collagen ispresent at a concentration of about 0.5 mg/ml to about 200 mg/ml. Inother embodiments, the collagen is present at a concentration of about0.5 mg/ml to about 100 mg/ml. In yet other embodiments, the collagen ispresent at a concentration of about 1 mg/ml to about 5 mg/ml. In stillyet other embodiments, the collagen is present at a concentration ofabout 2 mg/ml to about 5 mg/ml. In some embodiments, the collagen ispresent at a concentration of about 3.5 mg/ml. In other embodiments, thecollagen is present at a concentration of about 4 mg/ml to about 10mg/ml. In yet other embodiments, the collagen is present at aconcentration of about 5 mg/ml. In some embodiments, the collagen ispresent at a concentration of about 10 mg/ml to about 20 mg/ml. In otherembodiments, the collagen is present at a concentration of about 12mg/ml. In yet other embodiments, the collagen is present at aconcentration of about 20 mg/ml to about 30 mg/ml. In some embodiments,the collagen is present at a concentration of about 24 mg/ml. In someembodiments, the collagen is present at a concentration of about 500mg/ml. In other embodiments, the collagen is present at a concentrationof about 400 mg/ml. In yet other embodiments, the collagen is present ata concentration of about 300 mg/ml. In other embodiments, the collagenis present at a concentration of about 200 mg/ml. In yet otherembodiments, the collagen is present at a concentration of about 100mg/ml. In other embodiments, the collagen is present at a concentrationof about 75 mg/ml. In yet other embodiments, the collagen is present ata concentration of about 50 mg/ml.

In the embodiments described herein, the synthetic collagen-fibrilmatrices can have an oligomer content quantified by average polymermolecular weight (AMW). As described herein, modulation of AMW canaffect polymerization kinetics, fibril microstructure, molecularproperties, and fibril architecture of the matrices, for example,interfibril branching, pore size, and mechanical integrity (e.g., matrixstiffness). In another embodiment, the oligomer content of thepre-matrix composition, as quantified by average polymer molecularweight, positively correlates with matrix stiffness.

In some embodiments, a non-oligomeric soluble collagen included in thepre-matrix composition is reduced collagen. As used herein “reducedcollagen” means collagen that is reduced in vitro to eliminate orsubstantially reduce reactive aldehydes. For example, collagen may bereduced in vitro by treatment of collagen with a reducing agent (e.g.,sodium borohydride).

In accordance with certain aspects of the present disclosure, acollagen-based therapeutic delivery device comprises a syntheticcollagen-fibril matrix adapted for delivery of an active agent. Theincorporation of an active agent may be achieved by several methodsincluding, but not necessarily limited to, admixing the agent withsoluble collagen-fibril matrix building blocks in a pre-matrixcomposition prior to polymerization, exposing an already-formedsynthetic collagen-fibril material with an active agent followingpolymerization, and covalently attaching an active agent to a solublecollagen-fibril matrix building block and polymerizing the modifiedcollagen building block, either alone or in the presence of unmodifiedcollagen-fibril matrix building blocks. It should be understood andappreciated herein that the method of incorporating an active agent inthe synthetic collagen-fibril matrix may vary based on the active agent.It is further recognized that the method of incorporating a first activeagent in the collagen-fibril matrix may be the same or different fromthe method of incorporating a second active agent in the collagen-fibrilmatrix. It should also be understood and appreciated herein that theterms “first,” “second,” and “third” as applied to an active agent, areintended to allow distinctions between different active agents and donot necessarily convey a required chronological characteristic or order.

The purity of a collagen-fibril matrix in accordance with the teachingsof the present disclosure may be evaluated by any means known in the artincluding, but not limited to, SDS-PAGE either on the collagen polymerdirectly or after specific enzymatic (bacterial collagenase) or chemical(cyanogen bromide) cleavage, peptide mapping, amino-terminal sequencing,and non-collagenous impurity assays. Methods of characterizingcharacteristics of a collagen-fibril matrix include, but are not limitedto, cation-exchange HPLC, natural fluorescence, LC/MS, MS, dynamic lightscattering, size-exclusion chromatography, viscosity measurements,circular dichroism, differential scanning calorimetry, trypsinsusceptibility, impurity profiling, TEM∞ SEM∞ cryo-SEM∞ confocalmicroscopy, multiphoton microscopy and atomic force microscopy.

In accordance with certain aspects herein, qualitative and quantitativemicrostructural characteristics of a collagen-fibril matrix can bedetermined by environmental or cryostage scanning electron microscopy,transmission electron microscopy, confocal microscopy, second harmonicgeneration multi-photon microscopy. Tensile, compressive andviscoelastic properties can be determined by rheometry or tensiletesting. All of these methods are known in the art or are furtherdescribed in U.S. patent application Ser. No. 11/435,635 (published Nov.22, 2007, as Publication No. 2007/0269476 A1), U.S. patent applicationSer. No. 11/914,606 (published Jan. 8, 2009, as Publication No.2009/0011021 A1), U.S. patent application Ser. No. 12/300,951 (publishedJul. 9, 2009, as Publication No. 2009/0175922 A1), U.S. patentapplication Ser. No. 13/192,276 (published Feb. 2, 2012, as PublicationNo. 2012/0027732 A1), U.S. patent application Ser. No. 13/383,796(published May 10, 2012, as Publication No. 2012/0115222 A1), or aredescribed in Roeder et al., J. Biomech. Eng., vol. 124, pp. 214-222(2002), in Pizzo et al., J. Appl. Physiol., vol. 98, pp. 1-13 (2004),Fulzele et al., Eur. J. Pharm. Sci., vol. 20, pp. 53-61 (2003), Griffeyet al., J. Biomed. Mater. Res., vol. 58, pp. 10-15 (2001), Hunt et al.,Am. J. Surg., vol. 114, pp. 302-307 (1967), and Schilling et al.,Surgery, vol. 46, pp. 702-710 (1959), the disclosures of which are eachincorporated herein by reference in their entireties. Collagencharacteristics and methods of characterizing collagen characteristicsare discussed in ASTM International F3089-14, 2014 West ConshohockenPa., the disclosure of which is herein incorporated by reference in itsentirety.

In certain aspects of the present disclosure, the syntheticcollagen-fibril matrix exhibits a stiffness of at least 5 Pa. In anotherembodiment, the synthetic collagen-fibril matrix exhibits a stiffness ofbetween 5 Pa and 100 GPa. Stiffness may also be referred to as theelastic or linear modulus.

In some embodiments, the collagen-fibril matrix comprises a co-polymer,such collagen-fibril matrix being referred to herein as a “hybridcollagen-fibril” matrix. In one embodiment, the co-polymer comprises apolymerization product of a mixture of one or more types of solublecollagen building blocks with one or more synthetic or naturalnon-collagen molecule consisting of individual chemical moieties, whichmay be the different or the same. As used herein, the term “co-polymer”refers to individual chemical moieties that are joined end-to-end toform a linear molecule, as well as individual chemical moieties joinedtogether in the form of a branched (e.g., a “multi-arm” or“star-shaped”) structure. In alternative embodiments, the co-polymercomprises a copolymers that is obtained by copolymerization of twomonomer species, obtained from three monomers species (“terpolymers”),obtained from four monomers species (“quaterpolymers”) or obtained frommore than four monomer species. The present disclosure contemplatesembodiments of a hybrid collagen-fibril matrix that include collagenbuilding blocks associated with non-collagen molecules within thefibrils of the matrix (referred to herein as “hybrid fibrils”) and alsoembodiments of a hybrid collagen fibril matrix in which the noncollagenmolecules polymerize separately from the collagen fibrils to produceseparate collagen fibrils and non-collagen polymers within the hybridcollagen-fibril matrix. In another embodiment, a hybrid collagen-fibrilmatrix is formed by providing a polymerized non-collagen polymer orcopolymer defining pores and interstitial spaces, introducing an aqueousfluid comprising soluble collagen-fibril matrix building blocks into theinterstitial spaces and polymerizing the building blocks to form acollagen-fibril matrix within the interstitial spaces.

In another aspect, the present disclosure is directed to pre-matrixcompositions that are formulated for subsequent polymerization toprovide insoluble synthetic collagen-fibril matrices and/orcollagen-based therapeutic delivery devices. Polymerization of apre-matrix composition to form a collagen-fibril matrix, can beaccomplished under controlled conditions, wherein the controlledconditions include, but are not limited do, pH, phosphate concentration,temperature, buffer composition, ionic strength, and composition andconcentration of the building blocks and other optional moleculespresent in a given pre-matrix composition, which can include bothcollagen molecules and non-collagenous molecules.

As used herein, the term “pre-matrix composition” refers to an aqueousfluid that includes soluble collagen-fibril matrix building blocks, thebuilding blocks including oligomers and optionally one or more type ofnon-oligomeric soluble collagen molecules, which building blocksdemonstrate a capacity to self-assemble or polymerize into higher orderstructures (macromolecular assemblies) in the absence of exogenouscross-linking agents. In one embodiment, the building blocks areselected based on their molecular composition and fibril-formingcapacity. In one embodiment, the non-oligomeric soluble collagenmolecules include one or both of telocollagen molecules andatelocollagen molecules.

In accordance with an illustrative embodiment of the present disclosure,a pre-matrix composition comprises a collagen polymer solutioncomprising different types of collagen polymer building blocks,including but not limited to oligomer, monomer/telocollagen (alsoreferred to as telomer) and atelocollagen (also referred to asatelomer). These building blocks, as shown in FIG. 1 , differ based ontheir intermolecular cross-link content, composition and cross-linkchemistries. Referring to FIG. 1 , (A) depicts an oligomer, (B)telocollagen and (C) an atelocollagen. Gray bars in FIG. 1 representstable, mature covalent cross-links.

In some embodiments, the building blocks are obtained by solubilizingcollagen from tissue and purifying the soluble collagen. For example,the building blocks can be prepared by utilizing acid-solubilizedcollagen and defined polymerization conditions that are controlled toyield three-dimensional collagen matrices with a range of controlledassembly kinetics (e.g., polymerization half-time), molecularcompositions, and fibril microstructure-mechanical properties, forexample, as described in U.S. patent application Ser. No. 11/435,635(published Nov. 22, 2007, as U.S. Publication No. 2007/0269476) and Ser.No. 11/903,326 (granted Dec. 27, 2011, as U.S. Pat. No. 8,084,055), eachincorporated herein by reference in its entirety. In one embodiment, thecollagen is Type I collagen. In certain aspects of the presentdisclosure, the collagen-fibril matrix building blocks have been removedfrom a source tissue. Optionally, the building blocks may be solubilizedfrom the tissue source, while in still other embodiments, the buildingblocks comprise synthetic collagen. In still other embodiments, thebuilding blocks comprise recombinant collagen.

In any of the various embodiments described herein, the collagencompositions of the present disclosure can be combined, prior to,during, or after polymerization, with nutrients, including minerals,amino acids, sugars, peptides, proteins, vitamins (such as ascorbicacid), or glycoproteins that facilitate hematopoietic stem cell culture,such as laminin and fibronectin, hyaluronic acid, or growth factors suchas platelet-derived growth factor, or transforming growth factor beta,and glucocorticoids such as dexamethasone. In other illustrativeembodiments, fibrillogenesis inhibitors, such as glycerol, glucose, orpolyhydroxylated compounds can be added prior to or duringpolymerization. In accordance with one embodiment, cells can be added tothe collagen or extracellular matrix components as the last step priorto the polymerization or after polymerization of the collagencompositions. In other illustrative embodiments, cross-linking agents,such as carbodiimides, aldehydes, lysyl-oxidase, N-hydroxysuccinimideesters, imidoesters, hydrazides, and maleimides, and the like can beadded before, during, or after polymerization.

A variety of techniques can be used to control the therapeutic releaseprofile (also referred to as the molecular release profile) of acollagen-based therapeutic delivery system described herein. In oneaspect of this disclosure the pre-matrix composition is modulated toachieve synthetic collagen-fibril materials with a broad range oftunable or customizable fibril microstructures, mechanical properties,and proteolytic degradabilities by the selection of various proportionsof oligomeric and non-oligomeric building blocks. The modulation of thesynthetic collagen-fiber matrix composition and the self-assemblyreaction conditions allow the fibril microstructure and associatedinterstitial fluid viscosity, mechanical properties and proteolyticdegradation to be regulated. The design specificity of the syntheticcollagen-fibril matrix allows for the selection of mechanophysicalconstraints and bioinstructive capacity.

In certain aspects of the therapeutic delivery devices, thecollagen-fibril matrix may be tuned such that the predeterminedoligomer:non-oligomeric soluble collagen molecule ratio supports burstrelease, sustained release or variable release of the active agent.Burst release is a rapid release of the active agent within a shorttimeframe that delivers a bolus type amount of the agent to the targetarea. Sustained release provides an ongoing release of the active agentat a steady rate for a predetermined period of time. It is envisionedthat a therapeutic delivery system can be formulated to provide an earlyphase, medium phase or late phase burst release of an active agent. Itis further recognized that different active agents or differentconcentrations of an active agent may be released in different phases.It is also recognized that a first active agent may be released in asustained release while a second active agent may be released in a burstrelease.

In certain aspects, the therapeutic delivery device comprises more thanone layer of synthetic collagen-fibril matrix, each being distinguishedby at least one physical property or active agent. The layers may begenerated, for example in a spherical fashion, a cylindrical fashion, aplanar fashion or other three-dimensional fashion where one layer ofsynthetic collagen-fibril matrix is completely or almost completelysurrounded by at least a second layer of synthetic collagen-fibrilmatrix, wherein the second layer of collagen matrix comprises at leastone different physical property or active agent from the firstcollagen-fibril matrix layer. Further the collagen-fibril matrix layersmay differ by selection of a predetermined oligomer:non-oligomericsoluble collagen molecule ratio, by selection of different activeagents, or by different concentrations of the active agent. In anotherembodiment, the therapeutic delivery device comprises gradients of oneor more of physical properties, soluble collagen molecules and activeagents.

In addition, following formation of a synthetic collagen-fibril matrixby polymerization, the matrix can be further processed, for example byunconfined or confined compression, to achieve higher-density materialswith tissue-like consistency, handling and mechanical properties.Examples of compression processing options are described in U.S.Published Application No. 2015/0105323, the contents of which are herebyincorporated herein by reference.

In accordance with certain aspects of the present disclosure, asynthetic collagen-fibril matrix may be compressed to form a gradient ofat least one physical property. As used herein, the term “compressed”can refer to a reduction in size or an increase in density when a forceis applied to the collagen-fibril matrix. For example, compression canbe achieved through various methods of applying force, such as, but notlimited to, confined compression, variable compression, physicalcompression, centrifugation, ultracentrifugation, evaporation oraspiration. Moreover, in accordance with certain illustrative aspectsherein, it should be understood and appreciated that compressing thecollagen-fibril matrix can form a gradient of at least one physicalproperty in the composition. As used herein, the term “physicalproperty” can refer to any property of the collagen compositions,including structural, mechanical, chemical, and kinetic properties.

In accordance with certain embodiments, the gradient is acompression-induced gradient. As used herein, the phrase“compression-induced gradient” refers to a gradient in thecollagen-fibril matrix that is provided as a result of the compressionto which the collagen-fibril matrix is subjected.

In some embodiments, the compression is a physical compression. As usedherein, the phrase “physical compression” refers to compression of acollagen-fibril matrix by applying force by physical means.

In other embodiments, the compression is a confined compression. As usedherein, the phrase “confined compression” refers to confinement of thecollagen-fibril matrix as it undergoes compression.

In yet other embodiments, the compression is a variable compression. Asused herein, the phrase “variable compression” refers to compression ofa collagen-fibril matrix by applying force in a non-linear fashion.

In still other embodiments, the compression is centrifugation. In someembodiments, the compression is ultracentrifugation. In yet otherembodiments, the compression is evaporation. In some embodiments, thecompression is aspiration. In certain embodiments, the aspiration isvacuum aspiration. In select embodiments, the compression is not plasticcompression because such plastic compression may be an extreme processin which nearly all of the fluid removable from collagen compositions isexcreted, and can reduce the cellular viability of the scaffolds anddamage the natural matrix architecture.

For embodiments in which the compression is a physical compression, thephysical compression can be performed in a chamber comprising anadjustable mold and platen. Typically, collagen-fibril matrix can beinserted into the mold and then subjected to compression.

Furthermore, the physical compression can be varied depending on theplacement of the porous platen within the mold. For example, the moldmay be adjustable so that porous polyethylene is positioned as part ofthe platen and/or along the walls or bottom of the sample mold. Thelocation of the porous polyethylene can define the resultant materialgradient in the collagen-fibril matrix. In some embodiments, thecompression is a physical force from at least one direction. In otherembodiments, the compression is a physical force from two or moredirections. In yet other embodiments, the compression is a physicalforce from three or more directions. In some embodiments, thecompression is a physical force from four or more directions.

Pursuant to certain aspects of the present disclosure, a syntheticcollagen-fibril matrix as described herein may be made under controlledconditions to obtain particular physical properties. For example, thecollagen-fibril matrices may have desired collagen fibril density, poresize (fibril-fibril branching), elastic modulus, tensile strain, tensilestress, linear modulus, compressive modulus, loss modulus, fibril areafraction, fibril volume fraction, collagen concentration, cell seedingdensity, shear storage modulus (G′ or elastic (solid-like) behavior),and phase angle delta (δ or the measure of the fluid (viscous)—to solid(elastic)—like behavior; δ equals 0° for Hookean solid and 90° forNewtonian fluid).

As used herein, a “modulus” can be an elastic or linear modulus (definedby the slope of the linear region of the stress-strain curve obtainedusing conventional mechanical testing protocols; i.e., stiffness), acompressive modulus, a loss modulus, or a shear storage modulus (e.g., astorage modulus). These terms are well-known to those skilled in theart. As used herein, a “fibril volume fraction” (i.e., fibril density)is defined as the percent area of the total area occupied by fibrils inthree dimensions.

A collagen-based therapeutic delivery device as described herein can beformed for subsequent implantation into a patient, such as, for example,as a tissue graft material, or can be formed in situ by injecting apre-matrix composition to a location in situ for subsequentpolymerization to form a collagen-based therapeutic delivery material insitu.

In some embodiments, the collagen-based therapeutic delivery device is amedical graft. An important consideration for medical grafts,particularly soft tissue grafts is the design of a graft that promotesgraft vascularization, and particularly one that allows for cellco-implantation and cell infiltration, that structurally andfunctionally supports cell growth, and that is able to fully integratewith the tissue physiologically. Additional important considerationsinclude that the graft should not impede the growth of regeneratingtissue and that its degradation should not leave behind any byproductsthat would adversely affect the cells involved in tissue regeneration.The collagen-based therapeutic delivery systems described herein notonly allow for cell co-implantation and cell infiltration, structurallyand functionally supports cell growth, and are able to fully integratewith the tissue physiologically by providing a strong porous frameworksuitable for cell infiltration and growth, but they also enable thedelivery of vascularization-promoting active agents to the site of thegraft to enhance vascularization following implant. The collagen-fibrilmatrix can be formed in any two- or three-dimensional shape byconducting polymerization in a mold having a desired shape and sizeand/or by post-polymerization processing.

In other embodiments, the collagen-based therapeutic delivery system canbe used in vitro. For example, in vitro use of the collagen-basedtherapeutic delivery systems of the present disclosure may be utilizedfor research purposes such as cell tissue culture, drug discovery, andchemical toxicity testing. In other embodiments, the collagen-basedtherapeutic delivery system can be used in vitro for generating complextissue and organ constructs, including vascularization, for restorationof damaged or dysfunctional organs or tissues.

In accordance with certain aspects herein, the collagen-fibril matricesmay include, but are not limited to, low density fibril matrices andhigh density fibril matrices. A low density fibril matrix may have acollagen concentration less than about 10 mg/ml, 9 mg/ml, 8 mg/ml, 7mg/ml, 6 mg/ml, 5 mg/ml, 4 mg/ml, 3 mg/ml, 2 mg/ml or 1 mg/ml. A highdensity fibril matrix may have a collagen concentration greater than 10mg/ml, 20 mg/ml, 30 mg/ml, and 40 mg/ml or higher. Applications suitablefor low density fibril matrices may include, but are not limited to, invitro 3D cell culture, injectable therapeutic delivery, and implantabletherapeutic delivery. Applications suitable for high density fibrilmatrices or tissue constructs may include, but are not limited to,surgical implants, sheets, fibrillar material, tissue valves, tissuegradients, articular cartilage and tissue-engineered medical products.

It should be understood and appreciated herein that the illustrativecollagen-based therapeutic delivery systems of the present disclosurecan be used in human and veterinary medicine in both experimental and invivo conditions. Envisioned uses of the illustrative therapeuticdelivery systems in accordance with the teachings of the presentdisclosure include, but are not limited to, as hemostatic agents, asmissing tissue substitutes or replacements, as skin equivalents, and asmatrices for tissue augmentation. The desired physical characteristicsof the collagen-fiber matrix for use in different applications maydiffer depending on the application and the active agent.

Various examples demonstrating preparation and testing of compositions,processes and methods of the present disclosure are described in thefollowing examples. These examples are illustrative only and are notintended to limit or preclude other embodiments of the presentdisclosure. Moreover, it should be understood and appreciated hereinthat in order to measure and compare molecular release kinetics fromvarious collagen-fibril materials in the presence and absence ofcollagenase, an in vitro model system was designed to measure releasekinetics from collagen fibril materials by subjecting the polymerized 3Dcollagen matrix system with admixed FITC-Dextran molecules tocollagenase or IX PBS. In particular, the in vitro model system involvedadmixing of FITC-Dextrans of various sizes ranging from 10 kDa to 2 MDawithin polymerizable collagens, and then establishing a computationalmodel to predict release kinetics for various sized molecules based onknown diffusion coefficients for oligomer matrices. The designedexperimental model system was then used to define and comparesize-dependent molecular release kinetics for FITC-Dextrans withinlow-density matrices prepared with standardized collagen oligomers andcommercial monomeric collagen.

As used herein, the term “mammalian” refers to any species belonging tothe class Mammalia including, but not limited to, humans, cows, pigs,dogs, horses or cats.

As used herein, the term “mammalian tissue” refers to any tissueincluding, but not limited to, skin, muscle, tendons or fibrousconnecting tissue found in mammals.

As used herein, the term “diffusion” refers to the random thermal motionof atoms, molecules, clusters of atoms, etc. in gases, liquids, and somesolids.

As used herein, the term “fibrillogenesis” refers to the process oftropocollagen monomers assembling into mature fibrils and associatedfibril-network structures.

As used herein, the term “gel” refers to a three-dimensional networkstructure arising from intermolecular polymer chain interactions.

As used herein, the term “permeability” refers to a measure of theability of porous materials to transmit fluids; the rate of flow of aliquid through porous material.

As used herein, the term “recombinant collagen protein/peptide” refersto a collagen or collagen-like polypeptide produced by recombinantmethods, such as, but not limited to by expression of a nucleotidesequence encoding, the protein or peptide in a microorganism, insect,plant or animal host. Such compositions often comprise Gly-X-Y tripletswhere Gly is the amino acid glycine and X and Y can be the same ordifferent, are often proline or hydroxyproline, but can be any knownamino acid.

As used herein, the term “self-assembly” refers to the process by whicha complex macromolecule (for example collagen) or a supramolecularsystem (for example a virus) spontaneously assembles itself from itscomponents.

As used herein, the term “solution” refers to a type of homogenousmixture composed of only one phase. In such a mixture a “solute” is asubstance dissolved in another substance, known as a “solvent”.

As used herein, the term “stiffness” is a general term describing theextent to which a material resists deformation in response to an appliedforce; specific measures of stiffness depend upon the material loadingformat (for example, tension, compression, shear, bending).

As used herein, the term “degradation” refers to a change in chemical,physical or molecular structure or appearance (for example, grossmorphology) of material; degradation of collagen under physiologicalconditions involves site-specific cleavage within the central triplehelical region by proteolytic enzymes known as collagenases.Collagenases are members of the larger family of proteases known asmatrix metalloproteases.

As used herein, the term “solubility” refers to a measure of the extentto which a material can be dissolved; in the context of collagenpolymers, solubility refers to collagen molecules (partial, full ormultiples) or peptides in a solution; further qualification ofsolubility may include “acid-soluble” and “neutral salt-soluble” whichdescribe compositions that are soluble in dilute acids and neutral saltsolutions, respectively.

As will be appreciated from the above descriptions, taken together withthe Examples provided below, the present specification discloses a widevariety of forms and embodiments, some examples of which are describedas follows:

In one form, the present disclosure provides a collagen-basedtherapeutic delivery device that includes an insoluble syntheticcollagen-fibril matrix comprising a polymerization product of solubleoligomeric collagen or a polymerization product of a mixture of solubleoligomeric collagen with one or more type of non-oligomeric solublecollagen molecules; and a first active agent dispersed throughout thecollagen-fibril matrix or within a portion of the collagen-fibrilmatrix. Also provided are the following embodiments:

(1) Any of the embodiments disclosed herein wherein the collagen-fibrilmatrix exhibits a stiffness of at least 5 Pa.

(2) Any of the embodiments disclosed herein wherein the collagen-fibrilmatrix comprises type I collagen.

(3) Any of the embodiments disclosed herein wherein the one or moreother type of non-oligomeric soluble collagen molecules comprises one orboth of soluble telocollagen molecules and soluble atelocollagenmolecules.

(4) Any of the embodiments disclosed herein wherein the collagen-basedtherapeutic delivery device is a tissue graft.

(5) Any of the embodiments disclosed herein wherein the collagen-basedtherapeutic delivery device is lyophilized.

(6) Any of the embodiments disclosed herein wherein the collagen-fibrilmatrix comprises a polymerization product of a mixture of solubleoligomeric collagen with one or more type of non-oligomeric solublecollagen molecules and wherein the oligomeric collagen andnon-oligomeric soluble collagen molecules are in a ratio within a rangeselected from the group consisting of 0:100 to 5:95, 5:95 to 10:90,10:90 to 15:85, 15:85 to 20:80, 20:80 to 25:75, 25:75 to 50:50, 50:50 to75:25 and 75:25 to 100:0.

(7) Any of the embodiments disclosed herein, further comprising a secondactive agent dispersed throughout the collagen-fibril matrix or within aportion of the collagen-fibril matrix.

(8) Any of the embodiments disclosed herein wherein each of the firstand second active agents is a growth factor or a drug.

(9) Any of the embodiments disclosed herein wherein the collagen-fibrilmatrix includes a first portion having a first density and a secondportion having a second density; wherein the first density is differentthan the second density.

(10) Any of the embodiments disclosed herein wherein the collagen-fibrilmatrix includes a first portion having dispersed therein the firstactive agent and a second portion having dispersed therein a secondactive agent, wherein the therapeutic delivery device exhibits a firstrelease profile for the first active agent and a second release profilefor the second active agent, and wherein the first release profile isdifferent than the second release profile.

In another form, the present disclosure provides a method for making atherapeutic delivery device that includes (i) forming an aqueoussolution comprising a first quantity of soluble collagen-fibril buildingblocks; (ii) causing the building blocks to polymerize by self-assembly,thereby forming an insoluble synthetic collagen-fibril matrix; and (iii)either (a) including a second quantity of an active agent in the aqueoussolution whereby said causing forms the insoluble syntheticcollagen-fibril matrix having the active agent dispersed therein or (b)contacting the insoluble synthetic collagen-fibril matrix with thesecond quantity of the active agent to form a collagen-fibril matrixhaving the active agent dispersed therein; wherein the first quantity ofbuilding blocks comprises soluble oligomeric collagen molecules. Alsoprovided are the following embodiments:

(1) Any of the embodiments disclosed herein wherein the collagen-fibrilmatrix exhibits a stiffness of at least 5 Pa.

(2) Any of the embodiments disclosed herein wherein the first quantityof building blocks further comprises soluble non-oligomeric collagenmolecules.

(3) Any of the embodiments disclosed herein, further comprisingcompressing the insoluble synthetic collagen-fibril matrix to form acondensed insoluble synthetic collagen-fibril matrix.

(4) Any of the embodiments disclosed herein wherein said compressingcomprises subjecting the insoluble synthetic collagen-fibril matrix toconfined compression.

(5) Any of the embodiments disclosed herein, further comprising, aftersaid causing, lyophilizing the insoluble synthetic collagen-fibrilmatrix.

In still another form, the present disclosure provides a pre-matrixcomposition that comprises an aqueous solution including a firstquantity of soluble collagen-fibril building blocks and a secondquantity of an active agent in the aqueous solution, wherein the firstquantity of soluble collagen-fibril building blocks includes solubleoligomeric collagen or a mixture of soluble oligomeric collagen with oneor more other type of non-oligomeric soluble collagen molecules, whereinthe building blocks are operable to self-assemble into a macromolecularinsoluble synthetic collagen-fibril matrix having a stiffness of atleast 5 Pa in the absence of an exogenous cross-linking agent. Alsoprovided are the following embodiments:

(1) Any of the embodiments disclosed herein wherein the one or moreother type of non-oligomeric soluble collagen molecules comprises one orboth of soluble telocollagen molecules and soluble atelocollagenmolecules.

(2) Any of the embodiments disclosed herein wherein the oligomericcollagen comprises type I collagen.

(3) Any of the embodiments disclosed herein wherein the active agent isa growth factor or a drug.

(4) Any of the embodiments disclosed herein wherein the active agent iscovalently attached to one or more of the building blocks.

(5) Any of the embodiments disclosed herein wherein the pre-matrixcomposition comprises the oligomeric collagen and non-oligomeric solublecollagen molecules in a ratio within a range selected from the groupconsisting of 0:100 to 5:95, 5:95 to 10:90, 10:90 to 15:85, 15:85 to20:80, 20:80 to 25:75, 25:75 to 50:50, 50:50 to 75:25 and 75:25 to100:0.

(6) Any of the embodiments disclosed herein wherein the pre-matrixcomposition is capable of being modulated to achieve a nonsolublesynthetic collagen-fibril matrix that exhibits an optimized active agentrelease profile for the active agent.

In yet another form, the present disclosure provides a method fordelivering an active agent, that includes positioning at an in situposition (i) a pre-matrix composition comprising an aqueous solutionincluding a first quantity of soluble collagen-fibril building blocksand a second quantity of an active agent in the aqueous solution,wherein the first quantity of soluble collagen-fibril building blocksincludes soluble oligomeric collagen or a mixture of soluble oligomericcollagen with one or more type of soluble non-oligomeric collagenmolecules, wherein the building blocks are operable to self-assembleinto an insoluble synthetic macromolecular collagen-fibril matrix havinga stiffness of at least 5 Pa in situ in the absence of an exogenouscross-linking agent; or (ii) a collagen-based therapeutic deliverydevice comprising an insoluble synthetic collagen-fibril matrixcomprising a polymerization product of soluble oligomeric collagen or apolymerization product of a mixture of soluble oligomeric collagen withone or more type of soluble non-oligomeric collagen molecules; and afirst active agent dispersed throughout the collagen-fibril matrix orwithin a portion of the collagen-fibril matrix; wherein thecollagen-fibril matrix exhibits a stiffness of at least 5 Pa.

Example 1: Formulation of Collagen-Fibril Delivery Devices

Soluble collagen-fibril building blocks including oligomer,telocollagen, and atelocollagen (or oligomer, telomer, and atelomer)were derived from the dermis of market weight pigs. Oligomerformulations were prepared using an acid solubilization method thatpreferentially extracts oligomers, which represent aggregates ofcollagen molecules (e.g., trimer) covalently connected by a naturalintermolecular crosslink. Telomer formulations were prepared using acidsolubilization followed by a salt precipitation technique. Saltsolutions were used to selectively isolate collagen molecules, whichwere not cross-linked or which contained immature acid labilecross-links, particularly since telomer and oligomer formulationscontain reactive aldehyde groups on the telopeptide ends. Finally,atelocollagen formulations were prepared through a digestion techniquein which pepsin enzymatically cleaves the telopeptide ends from the N-and C-terminus of the collagen molecule. These soluble collagen-fibrilbuilding blocks are standardized and quality controlled based upon theirmolecular composition and polymerization (collagen-fibril formation)capacity. For comparison to commercial grade collagen, acid solubilizedtype I collagen harvested from rat tails was purchased from BDBiosciences (Bedford, Mass., referenced as BD-rat tail collagen,BD-RTC).

For preparation of collagen-fibril delivery devices from various acidsolubilized collagen solutions, the collagens were further diluted in0.01N HCl and further neutralized with phosphate buffered saline (PBS,10×, pH 7.4) and 0.1N sodium hydroxide (NaOH) to achieve neutral pH(7.4) and a final collagen concentration of 3 mg/ml. For formulatingmatrices with molecules admixed freely in it, FITC-Dextran of fourdifferent sizes (i.e., 10, 40, 500 and 2 MDa) were loaded to mimicdifferent sized therapeutic molecules. Here, FITC-Dextrans (10 kDa, 40kDa, 500 kDa, or 2 MDa, from Invitrogen, Eugene, Oreg.) were solubilizedin 10×PBS to yield desired final concentration within polymerizedmatrices. FITC-Dextran enables straightforward quantification of releasekinetics based on fluorescence level detection from supernatant abovecollagen matrix system. All the neutralized solutions were polymerizedin 0.25 ml volume in a 48 well plate (Corning, USA), as shown in FIG. 2. The system allowed molecular release kinetics into 0.75 ml top bufferwhich was 1× PBS (pH 7.4) in the absence or presence of collagenase(type IV, from Worthington Biochemical Corporation, USA).

Example 2: Admixing FITC-Dextran has No Effect on Collagen-FibrilPolymerization Kinetics or Collagen-Fibril Matrix Visco-ElasticProperties

To confirm that the addition of FITC-Dextran had no effect oncollagen-fibril polymerization kinetics and collagen-fibril deliverydevice physical properties, oligomer matrices (3 mg/ml) were neutralizedin the absence and presence of FITC-Dextrans (10 KDa and 2 MDa, each ata final concentration of 1.0 mg/ml) as described in procedure above.Each neutralized solution was put on AR2000 rheometer adapted with astainless steel 40 mm diameter parallel plate geometry (TA Instruments,New Castle, Del.) for polymerization in contact with the steel plate.Time-dependent changes in viscoelastic properties (shear storage modulus(G′), shear loss modulus (G″), and tan of phase shift delta (δ)) duringpolymerization were monitored through oscillatory shear, using a timesweep procedure. Temperature of the rheometer plate was maintained at 4°C. for a period of 5 minutes to get the baseline of storage modulus ofneutralized collagen solutions with or without FITC-Dextran prior topolymerization. The temperature was then increased to 37° C. to inducepolymerization of matrices. Polymerization kinetics and viscoclasticproperties of matrices prepared with or without FITC-Dextran werecompared. Polymerization half time (Thalf) was calculated along with therate of polymerization, defined as the slope of polymerization curve foreach sample. Each formulation was tested in triplicate.

Results indicated that admixing FITC-Dextrans at 1 mg/ml did notsignificantly affect collagen-fibril polymerization kinetics or theviscoelastic properties for the resultant collagen-fibril matrix(p>0.05), as shown in FIG. 3 . In particular, FIG. 3A showspolymerization kinetics as measured by time-dependent changes in G′,while FIG. 3B shows associated final G′ values for 3 mg/ml oligomermatrices prepared in absence and presence of 10 KDa and 2000 KDaFITC-Dextrans (1 mg/ml).

Example 3: Predicting Time Required for Diffusion-Based Release ofVarious Sized FITC-Dextran Molecules from Collagen-Fibril Materials

To predict the diffusion-based release kinetics of various sizedFITC-Dextrans from collagen-fibril matrices as well as define reservoirsampling times, an established mathematical model for monolithicmatrices was adapted. The mathematical model was based on Fick's secondlaw of diffusion for a slab matrix geometry with homogeneous initialdrug distribution within the collagen matrix system and an associatedsupernatant “sink.”

Equations used for predictive modeling of short-time and long-timerelease were:

Short-time:

$\frac{M_{t}}{M_{\infty}} = {4\left( \frac{Dt}{\pi L^{2}} \right)^{\frac{1}{2}}}$

Long-time:

$\frac{M_{t}}{M_{\infty}} = {1 - {\frac{8}{\pi^{2}}{\exp\left( {- \frac{\pi^{2}{Dt}}{L^{2}}} \right)}}}$

Here, M_(t) and M_(∞) denote the cumulative amounts of drug released attime t and at infinite time respectively; D is the diffusion coefficientof the drug within the system, and L represents the total thickness ofthe matrix. Length values for collagen-fibril matrices were 2.61 mm asdefined by our experimental system. Diffusion coefficient values (D)used for 10 KDa, 40 KDa, 500 KDa and 2 MDa FITC-Dextrans were 1.09 E-10,4.8 E-11, 2.52 E-11, and 1.76 E-11 (m²/sec) based upon previouspublished values for 3 mg/ml oligomeric collagen matrices.

Results obtained showed that release rates decreased while T50% ofrelease (Time required for 50% of cumulative release) increased as theFITC-dextran size increased, as demonstrated in FIG. 4 . Suchsize-dependent release kinetics might be expected as the diffusioncoefficient (D) of FITC-Dextrans decreases with increasing molecularsize.

Example 4: Comparison of Size-Dependent Molecular Release Kinetics forLow-Density (3 mg/ml) Oligomer and Commercial Monomer Collagen-FibrilMatrices in the Presence and Absence of Collagenase

To further validate and demonstrate the utility of the designedexperimental model for defining molecular release kinetics ofcollagen-fibril matrices, 10 KDa, 40 KDa, 500 KDa and 2 MDaFITC-Dextrans were admixed in oligomer and commercial monomer matricesand their release was compared from the designed in vitro experimentalmodel system. Matrices (250 μl each) were prepared in 48 well pates with750 μl reservoir of PBS (1×, pH 7.4) on top to induce diffusion-basedrelease. To simulate release kinetics based on both diffusion andproteolytic degradation of collagen-matrices, a subset of experimentswere conducted in the presence of 125 U/ml collagenase from ClostridiumHistolyticum (Worthington Biochem Corporation, USA). It should be notedthat the reservoir buffer choice made in accordance with the presentsystem allowed the following to be investigated 1) the effect ofcollagen microstructure alone on molecular release, when 1× PBS was used(absence of collagenase condition); and 2) the effect of collagenmicrostructure along with its proteolytic degradability on molecularrelease, when 125 U/ml collagenase was used (presence of collagenasecondition).

At various time points, the supernatant was removed and replaced withfresh PBS or collagenase according to the system. FITC-dextran withinthe supernatant then was determined spectrofluorometrically (MolecularDevices Spectramax M5) at excitation and emission wavelengths of 493 and530 nm, respectively. This process was repeated until RelativeFluorescence Units from supernatant of wells matched baselinefluorescence (PBS plus/minus collagenase containing no FITC-Dextran),indicating completion of the FITC-Dextran release. All fluorescencevalues were normalized to maximum total fluorescence intensity and %cumulative release was plotted versus time. Additional effects ofcollagenase levels on release kinetic curves for 10 KDa and 2 MDaFITC-Dextran can be seen in the graphical representation of FIG. 15 . Inparticular, low-density polymerized oligomer matrices (3 mg/ml)containing 10 KDa or 2 MDa FITC-dextran were subjected to variouscollagenase levels and release kinetics measured. In addition, FIG. 16depicts illustrative graphs showing the initial rates of release andT50% curves calculated from the release kinetics curves for the variouscollagenase concentrations.

Referring now to FIG. 5 , size dependent molecular release was observedwith polymerizable oligomer matrices but not commercial BD rat tailmatrices. More particularly, FITC-Dextrans with molecular sizes of 10kDa (X), 40 kDa (□), 500 kDa (Δ), and 2 MDa (◯) were polymerized at 0.5mg/ml within oligomer and commercial monomer (BD rat tail) matrices (3mg/ml). For oligomer (A) and commercial monomer (D) collagen matrices,time-dependent release profiles were monitored spectrofluorometrically,and initial release rate (mean±SD; n=3; B,E) and T50% (mean±SD; n=3;C,F) quantified. For each panel, letters indicate statisticallydifferent experimental groups as determined by Tukey-Kramer range test(p<0.05). When 1× PBS buffer was used to investigate effect of collagenmicrostructure on molecular release, results showed that oligomermatrices showed release profiles that were dependent on the size ofFITC-Dextrans (FIG. 5A). In contrast, such size dependent molecularrelease was not observed with commercial monomer matrices prepared atthe same concentration (FIG. 5D). The quantification of initial releaserates from oligomer (B) showed a trend of decreasing release rate asFITC-Dextran size became larger (10 KDa to 2 MDa) and an increasingtrend for T50% (C). These trends were consistent with those predicted bythe computational model. No such size-dependent trends were observed inthe release rates or T50% for commercial monomer matrices. The resultsshowed that the microstructure of oligomer collagen-fibril matricesprovided improved control of FITC-Dextran release kinetics, compared tothat of commercial BD rat tail collagen monomer matrices.

Referring to FIG. 6 , oligomer matrices in the presence of collagenasewere found to maintain size dependent and sustained release trends,while commercial monomer (BD rat tail) matrices did not. In particular,FITC-Dextrans with molecular sizes of 10 kDa (X), 40 kDa (□), 500 kDa(Δ), and 2 MDa (◯) were polymerized at 0.5 mg/ml within oligomer andcommercial monomer matrices (3 mg/ml). For oligomer (A) and commercialmonomer (D) matrices, time-dependent release profiles were monitoredspectrofluorometrically in the presence of collagenase (125 U/ml), andinitial release rate (mean±SD; n=3; B,E) and T50% (mean±SD; n=3; C,F)were quantified. For each panel, letters indicate statisticallydifferent experimental groups as determined by Tukey-Kramer range test(p<0.05). When 125 U/ml collagenase was used to investigate the effectof proteolytic degradability in addition to collagen microstructure onthe FITC-Dextran release kinetics, oligomer matrices maintained sizedependent and sustained release profiles (FIG. 6A), while commercialmonomer collagen matrices showed more rapid “burst” release for allmolecular sizes tested for a given time period (FIG. 6D). Thequantification of initial slopes of release profiles gave significantlyhigher rates of release and significantly lesser T50% for commercialmonomer matrices compared to the oligomer matrices (p<0.05), indicatingthe rapid proteolytic degradability and inability of commercial monomermatrices to support molecular release for extended period of time.

Thus, results indicated successful formation of a multifunctionalbiograft material that was experimentally tested for release of varioussized FITC-Dextran molecules in vitro. The designed experimental modelallowed the effect of the collagen-fibril microstructure to beinvestigated alone, as well as both collagen-fibril microstructure andproteolytic degradability on molecular release.

After establishing the functionality and robustness of the illustrativecollagen-fibril matrix system for delivery of different sizes ofFITC-Dextran molecules, the collagen biograft system can then bemodulated to make it tunable for release. It should be understood andappreciated herein that molecular release can be affected by two keyparameters, namely 1) the collagen fibril microstructure, and 2) itsproteolytic degradability. As such, it was an objective to incorporateapproaches for modulating these two parameters to customize releasekinetics from collagen-fibril materials. It was believed that by usingdifferent interfibril branching capacity of collagen building blocks andby altering the collagen fibril density, molecular release kinetics atsuprafibrillar level of assembly can be tuned.

It was proposed to characterize molecular release from low-density (3mg/ml) collagen-fibril materials, and then to modulate molecular releasefrom these collagen-fibril materials by combining two types of buildingblocks in collagen-fibril matrices. Thereafter, it was proposed toincrease collagen fibril-density to provide the molecular release forextended periods of time and to characterize release from thesehigh-density materials. To further modulate molecular release from thesehigh-density collagen-fibril materials, it was proposed to combineoligomer and telocollagen; and oligomer and atelocollagen blocks indifferent ratios as a means to modulate proteolytic degradability.

In terms of characterizing molecular release from low-densitycollagen-fibril materials, it should be understood and appreciatedherein that collagen precursors, telocollagens, oligomers andatelocollagens, differ in their intermolecular cross-link composition.It has been previously shown that collagen precursors provideindependent control of mechanical and transport properties of collagenmatrix. As such, it is therefore hypothesized that the matrices formedfrom these precursors would exhibit different release kinetics based ontheir varying fibril microstructure, as well as varying proteolyticdegradability, at a matched concentration. In order to test this,proteolytic degradability of matrices were first studied, followed bycharacterization of molecular release from them in both absence andpresence of collagenase. It was also proposed to confirm the effect ofmicrostructure and proteolytic degradability on the release mechanism ofmatrices by fitting a Weibull function to the molecular release data.

In order to confirm the proteolytic degradability differences ofoligomer vs. telocollagen and atelocollagen matrices, an experiment wasperformed in which 3 mg/ml oligomer, telocollagen and atelocollagenmatrices were polymerized on rheometer plate at 4° C. for 5 minutes toget a baseline, and then the temperature was ramped up to 37° C. forpolymerization. After 30 min of polymerization, the matrices formed wereexposed to 5000 U/ml collagenase in an oscillatory shear-based, andstrain-controlled time sweep experiment and tan of phase shift angledelta was tracked with respect to time as shown in FIG. 7 . Absolute tandelta was plotted as a function of time and representative samples ofeach type of matrix are shown in this figure. Rise in tan delta value≥1indicated phase change from solid to liquid, indicating completeproteolytic degradation of material. First derivative of tan delta wasthen plotted against time to get an inflection peak, which was used tocalculate the total degradation time for matrices, after subtracting thefirst 30 minutes of polymerization time. Data analysis showed thatdegradation time required for matrices was significantly different foroligomer, telocollagen and atelocollagen matrices (p<0.05; N=3). Thematrices degraded in the order of atelocollagen (138.0 min), followed bytelocollagen (186.7 min), followed by oligomer (219.5 min) matrices,indicating that atelocollagen and oligomer matrices represented theextreme ends of the obtained proteolytic degradation spectrum. Theresults suggested that the use of atelocollagen, telocollagen, andoligomer as collagen building block can offer an opportunity to tunerelease kinetics of contained drug molecules owing to differentproteolytic degradability and different fibril microstructureproperties.

Based on the fibril microstructure and proteolytic degradationdifferences between various collagen building blocks, as reportedpreviously, it was hypothesized that matrices formed from differentcollagen precursors will deliver molecules with different releasekinetics. In order to test this hypothesis, 3 mg/ml polymerized matricesfrom oligomer, telocollagen and atelocollagen building blocks wereformulated, containing 10 kDa and 2 MDa FITC-Dextran, at 0.25 mg/mlfinal concentration. The formulation was performed according to theprocedure described above with respect to the formulation ofcollagen-fibril delivery devices (Example 1). Release kinetics fromformulated delivery systems were then measured under two conditions: 1)in absence of collagenase, to investigate effect of microstructure onrelease kinetics (diffusion only); and 2) in presence of 125 U/mlcollagenase, to investigate effect of both microstructure andproteolytic degradability on the release kinetics(diffusion+degradation).

Referring now to FIG. 8 , a graphical representation illustrating thatthe molecular release profiles are dependent upon the collagen polymerbuilding blocks is shown. In particular, FITC-Dextrans with molecularsizes of 10 kDa and 2 MDa were polymerized within oligomer,telocollagen, and atelocollagen matrices (3 mg/ml). Time-dependentrelease profiles were monitored spectrofluorometrically in the absence(A,B) and presence (C,D) of collagenase (125 U/ml) and initial releaserate (mean±SD; n=3; A,C) and T50% (mean±SD; n=3; B,D) quantified. Foreach panel, letters indicate statistically different experimental groupsas determined by Tukey-Kramer range test. Results showed that in absenceof collagenase, microstructure-based differences in the oligomer,telocollagen, and atelocollagen affected release kinetics parameters(FIGS. 8A and 8B). It was clear that the atelocollagen and oligomer werethe building blocks that formed extreme ends of obtained releasespectrum again.

In presence of collagenase, the rate of release was enhanced, due toproteolytic degradability in addition to collagen microstructure-baseddiffusion, both contributing to the molecular release. Interestingly,the proteolytic degradation as measured in the presence of collagenaseemphasized differences in telocollagen and oligomer matrices in additionto those between oligomer and atelocollagen matrices. As seen in FIGS.8C and 8D, a progressive increase in initial release rate and aprogressive decrease in T50% values (p<0.05) was obtained for oligomer,telocollagen, and atelocollagen matrices in the presence of collagenase.

This experiment showed that collagen-fibril matrices composed ofdifferent building blocks demonstrated different molecular releaseprofiles, due to the different fibril microstructure and differentproteolytic degradation of matrices.

Example 5: Deciphering Effect of Different Collagen Building Blocks onRelease Mechanism of Molecules Using Weibull-Function

There are several empirical models available for simulating drug releasefrom polymer matrices. Although the power law model has been extensivelyused, it is confined for the description of the first 60% of the releasecurve. The quality of the fit has been observed to be poor at longertime points where the cumulative release exceeds ˜60%. Weibull functionis another alternative that can be used for the description of releaseprofiles based on the empirical use of the Weibull function described bythe equation:

$\frac{M_{t}}{M_{\infty}} = {1 - {{\exp\left( {- {at}^{b}} \right)}.}}$

M_(t) is the mass of drug released at time t, M_(∞) is the mass of drugreleased at infinite time (assumed equal to the amount of drug added), adenotes a scale parameter of time dependency, while b describes theshape of the dissolution curve progression. Papadopoulou et al. provideda powerful link between the shape parameter b and the diffusionalmechanisms of the release, as shown in the Table 1 below (see,Papadopoulou V, Kosmidis K, Vlachou M, Macheras P: On the use of theWeibull function for the discernment of drug release mechanisms.International journal of pharmaceutics (2006) 309(1-2):44-50).

TABLE 1 Exponent b of Weibull function and mechanism of release bRelease mechanism-remarks b < 0.35 Not found in simulation and theexperimental results. May occur in highly disordered spaces muchdifferent than the percolation cluster. b~0.35-0.39 Diffusion in fractalsubstrate morphologically similar to the percolation cluster 0.39 < b <0.69 Diffusion in fractal or disordered substrate different from thepercolation cluster b~0.69-0.75 Diffusion in normal Euclidian space 0.75< b < 1   Diffusion in normal Euclidian substrate with contribution ofanother release mechanism b = 1   First order release obeying Fick'sfirst law of diffusion; the rate constant a controls the releasekinetics and the dimensionless solubility/dose ratio determines thefinal fraction of dose dissolved b > 1   Sigmoid curve indicative ofcomplex release mechanism

It was hypothesized that by fitting Weibull function to experimentallyobtained release curves obtained with different soluble collagen-fibrilbuilding blocks, the underlying release mechanisms could be deciphered.Furthermore, by using Weibull function-based simulation of releasekinetics in absence of collagenase condition, it should be validated howmicrostructure based differences alone caused by different interfibrilbranching capacity of collagen building blocks, affect their molecularrelease kinetics.

To this end, the Weibull function was incorporated into the experimentalrelease kinetics obtained from commercial monomer collagen (BD rattail), atelocollagen, telocollagen, and oligomer matrices admixed withvarious FITC-Dextran molecules. The Weibull function fit theexperimental data well, and the parameter values a and b along with R²and confidence intervals determined for parameters a and b from the fitsare given in Table 2 below.

Results show that commercial monomer (BD rat tail) matrices gave adiffusion based release mechanism, while oligomer and telocollagencollagen-fibril matrices showed a combined release mechanism (Fickiandiffusion and Case II transport) associated with them. Atelocollagenshowed completely different value of parameter b, perhaps indicatingdiffusional release mechanism as in highly disordered spaces. Thus,since Weibull function was fit to release kinetics in absence ofcollagenase alone, the results confirmed that collagen-fibrilmicrostructure affects molecular release.

TABLE 2 Weibull function based parameters a and b for release kineticsfrom collagen matrices formulated from different building blocks (N = 3for each matrix) BD Rat a b R square CI (a; b) 2 MDa 0.028348 0.5332560.918419  [0.0089, 0.4517; 0.04784, 0.6148] 500 kDa 0.021974 0.5335840.932005 [0.0107, 0.4728; 0.0332, 0.5943] 40 kDa 0.014174 0.6082310.944953 [0.0064, 0.5438; 0.0219, 0.6727] 10 kDa 0.017661 0.5859250.951242 [0.0089, 0.5279; 0.0263, 0.6439] Conclusion: Diffusion innormal Euclidian space for all molecular sizes Atelocollagen A b Rsquare CI (a; b) 2 MDa 0.17026 0.328441 0.840058 [0.0927, 0.2735;0.2478, 0.3834] 10 kDa 0.281443 0.279144 0.808817 [0.1668, 0.2296;0.3961, 0.3287] Conclusion: May occur in highly disordered spaces muchdifferent than the percolation cluster Telomer A b R square CI (a; b) 2MDa 0.001083 0.876103 0.954832 [0.0002, 0.7883; 0.0019, 0.9639] 500 kDa0.000405 0.984194 0.946009 [2.1507e−05, 0.8744; 0.0008, 1.0939]   40 kDa0.003291 0.778927 0.973534 [0.0016, 0.7193; 0.0050, 0.8386] 10 kDa0.008298 0.675238 0.973083 [0.0047, 0.6244; 0.0118, 0.7260] Conclusion:Diffusion in normal Euclidian substrate with contribution of anotherrelease mechanism for all molecular sizes except 10 kDa, which showsdiffusion in normal Euclidian space Oligomer A b R square CI (a; b) 2MDa 0.001157 0.870807 0.942191 [0.0002, 0.7715; 0.0021, 0.9701] 500 kDa0.000473 0.96695 0.951171 [5.3077e−05, 0.8640; 0.0009, 1.0699]   40 kDa0.002865 0.795155 0.964108 [0.0011, 0.7237; 0.0046, 0.8666] 10 kDa0.006484 0.70258 0.963082 [0.0030, 0.6401; 0.0099, 0.7650] Conclusion:Diffusion in normal Euclidian substrate with contribution of anotherrelease mechanism for all molecular sizes except 10 kDa, which showsdiffusion in normal Euclidian space

Example 6: Effect of oligomer:atelocollagen ratio of polymerizationkinetics

To determine if the oligomer:atelocollagen ratio had an effect oncollagen-fibril matrix polymerization kinetics, the matrices werepolymerized without FITC-Dextran on AR2000 Rheometer using the proceduredescribed in Example 2. Five different matrix combinations were preparedby combining soluble oligomer and atelocollagen building block in theratio of 0:100, 25:75, 50:50, 75:25, and 100:0 prior to polymerization.The resultant polymerization curves and calculated polymerization ratesand T50% are shown in FIG. 9 . In particular, time-dependent changes inshear-storage modulus were monitored as collagen formulations exhibitedsolution to matrix transition following an increase in temperature from4° C. to 37° C. The polymerization profiles (A) were used to quantifyinitial rate of polymerization (mean±SD, B) and half-polymerizationtimes (mean±SD, C) for N=3 of each matrix type.

It was observed from FIG. 9 that the different matrix combinationsshowed different polymerization profiles (p<0.05, N=3). Stiffness valuesof formed matrices increased as the percentage of oligomer increased(FIG. 9A). Rate of polymerization increased (FIG. 9B), and T50%decreased (FIG. 9C) with increasing oligomer content. Rapidpolymerization times (T50%<5 minutes) were observed for alloligomer:atelocollagen ratios except 0:100.

Interestingly, all the matrix combinations displayed rapidpolymerization (T50%<5 minutes), except the 0:100 oligomer:ateocollagenratio (pure atelocollagen) matrix (FIG. 9C). Rapid collagen-fibrilpolymerization is an important design feature. In clinical applications,it is necessary that injected collagen solutions polymerize quickly tocreate a solid matrix that will allow for appropriate matrix placementand molecular delivery in situ, a feature, not exhibited by manyconventional collagen formulations. These results indicated that withthe use of different combinations of oligomer and atelocollagen buildingblocks, one can prepare matrices with different stiffness whileretaining their potential to polymerize within 5 minutes (exception:100% atelocollagen matrix).

Example 7: Modulation of Matrix Release Kinetics by Varying theCompositional Ratio of Molecular Building Blocks Oligomer andAtelocollagen

The different microstructures and proteolytic degradation obtained formatrices with different combinations of oligomer: atelocollagen ratiosimplied that the modulation in matrix composition would translate intoits different agent release profiles. It was hypothesized thatmodulating the matrix composition using oligomer and atelocollagenbuilding blocks could tune molecular release kinetics of low-fibrildensity matrices (3 mg/ml). In order to test this, collagen-fibrilmatrices were formulated with combinations of oligomer: atelocollagen ina ratio of 0:100, 5:95, 10:90, 15:85, 20:80, 25:75, 50:50, 75:25 and100:0%. These matrices were admixed with 0.5 mg/ml of 1) 10 KDaFITC-Dextran molecules in one subset and 2) 2 MDa FITC-Dextran moleculesin another subset, and their release was studied in absence or presenceof 10 U/ml collagenase.

The results showed that low fibril-density (3 mg/ml) collagen matricesgave similar diffusional release profiles, (FIG. 10A for 10 kDa, and 10Cfor 2 MDa) but different collagenase-dependent release profiles (FIG.10B for 10 kDa, and 10D for 2 MDa). The release profiles also showedenhanced time for complete release of 2 MDa FITC-Dextran compared to 10KDa FITC-Dextran molecules (FIGS. 10C and D). This implied the tendencyof matrices to retain larger sized molecules for longer time withinthem, than the smaller sized molecules.

Example 8: Molecular Release from High-Density Collagen-Fibril Materials

It has been established previously that collagen-fibril densityincreases and pore size decreases with increasing soluble collagenbuilding block concentration. It is also known that molecular diffusionand proteolytic degradation decrease with increase collagen-fibrildensity. As such, it was hypothesized that the release from thesynthetic collagen-fibril matrices would be prolonged by increasing theconcentration of pre-mixed soluble collagen building blocks. In order totest this, the oligomer collagen-fibril matrices were prepared at low (3mg/ml) and high densities (15.6 mg/ml). Low-density matrices wereprepared as described previously. High-density matrices were prepared bysubjecting 3 mg/ml collagen-fibril matrices to confined compression toachieve 5.2× volume reduction. Cylinders of diameter 1.1 (thickness 2.6mm) were prepared from each matrix type and release kinetics compared.

Example 9: Creation of High-Density Collagen-Fibril Delivery Devices

It was desired to create collagen matrices with increased fibril densitywithout compromising their ability to support and induce cellinfiltration and tissue regeneration. To this end, it has been shownthat the oligomer matrices have an advantage in creating tissue-likematerials without reducing porosity to an extent that cells cannotinfiltrate it. Therefore, oligomer collagen matrices were densified as aproof-of-concept study. In particular, the effective oligomerconcentration, which translates to fibril density, of the polymerizedmatrices was increased by the method of confined compression developedby Harbin laboratory. A schematic of the process for creating ahigh-fibril density matrix via confined compression in accordance withthe teachings of the present invention is shown in FIG. 14 . Briefly,10.82 ml of soluble oligomer collagen (3 mg/ml) admixed with 2 MDa or 10kDa FITC-Dextran was pipetted into compression molds (2 cm width by 4 cmlength). The solutions were polymerized overnight at 37° C. to createcollagen-fibril matrices with a thickness of 13.52 mm. The polymerizedmatrices with admixed FITC-Dextran were then subjected to confinedcompression using porous polyethylene platen (50 micron pore) at 6mm/min to a final thickness 2.6 mm (2 cm width by 4 cm length). Finalconcentration of compressed collagen matrices was 15.6 mg/ml (5.2×compression). Cylinders of diameter 1.1 (thickness 2.6 mm) were preparedfrom low-density (3 mg/ml) and high-density (15.6 mg/ml) matrices.Molecular release profiles were measured in the presence of 50 U/mlcollagenase for triplicate samples of each matrix formulation.

Example 10: Validating the Extension of Molecular Release from DensifiedOligomeric Collagen

The formulated collagen matrices of altered density (3 mg/ml=low; and15.6 mg/ml=high) were compared for molecular release of 10 kDa and 2 MDaFITC-Dextran each, as shown in FIG. 11 . In particular, 10 kDa and 2 MDaFITC-Dextran was admixed in 3 mg/ml and 15.6 mg/ml oligomer matrices.Release kinetics for both matrices were measured upon exposure to 50U/ml collagenase. As expected, the difference between release profilesexhibited by low-density and high-density matrix formulation is enhancedfor 2 MDa FITC-Dextran (D) as compared to 10 kDa FITC-Dextran (A). Theinitial rate of release is significantly lower in high density matricescompared to low density matrices for both 10 KDa (B) and 2 MDa (E)FITC-Dextrans. The T50% of release is significantly higher in highdensity matrices than in low density matrices for both smaller sizeFITC-Dextran (10 kDa) and larger size (2 MDa) as seen in FIGS. 10E and10F. Results showed that FITC-Dextran release from compressed matriceswas significantly more prolonged compared to non-compressed samples forboth 10 KDa (FIG. 11A) and 2 MDa (FIG. 11D) FITC-Dextrans. A decrease ininitial rate of release and an increase in T50% values, was alsoobserved with higher collagen fibril density, for both 10 KDa (FIGS.11B, C) and 2 MDa (FIG. 11E, F) FITC-Dextrans. It was concluded fromthis experiment that densifying collagen matrices thus modulatesmolecular release from collagen-fibril matrices.

Example 11: Effect of Density of Oligomer Collagen-Fibril Matrices onRelease of 10 kDa and 2 MDa FITC-Dextran Release

To further define the how the density of oligomer collagen-fibrilmatrices affects agent release, 10 kDa and 2 MDa FITC-dextrans wereadmixed at a concentration of 0.25 mg/ml within matrices prepared overthe density range of 3 mg/ml to 40 mg/ml. Oligomer matrices prepared at3 mg/ml were prepared as previously described in absence of confinedcompression, while 20 mg/ml and 40 mg/ml matrices were prepared byapplication of confined compression to 4.05 mg/ml matrices. Briefly,10.39 ml and 20.78 ml of neutralized oligomer collagen (4.05 mg/ml) wasadmixed with 10 kDa or 2 MDa FITC-dextran (0.25 mg/ml) and pipetted intocompression molds (2 cm width by 4 cm length). The solutions werepolymerized overnight at 37° C. to form matrices of 1.3 cm and 2.6 cmthickness respectively. These matrices were then subjected to confinedcompression using a porous polyethylene platen to final thickness of0.26 cm to achieve a 4.84× (20 mg/ml) and 9.88× (40 mg/ml)densification. Cylinders of diameter 1.1 (thickness 0.263 cm) wereprepared from all matrices and used to measure release kinetics in thepresence or absence of collagenase (10 U/ml). All measurements were madeon triplicate samples. Release profiles were plotted and analyzed fortime required for 50% cumulative release (T50%), which was calculatedbased on Weibull fit. The resultant Weibull parameters were used todefine the release mechanism.

The oligomer collagen-fibril matrices showed a density-dependent effecton molecular release of both 10 kDa and 2 MDa FITC-dextrans in thepresence of 50 U/ml collagenase as shown in FIGS. 17 and 19 . For bothFITC-dextrans, an increase in T50% was observed with increased density.As expected, the dynamic range of density-dependent release was greatestfor 2 MDa FITC-dextran. In addition, 10 kDa FITC-dextran showeddecreased T50% values compared 2 MDa FITC-dextran for all matricestested. Interestingly, Weibull analysis indicated that the 10 kDarelease mechanisms for both 3 mg/ml and 20 mg/ml oligomer matricesinvolved both diffusion and degradation. On the other hand, theincreased resistance to collagenase degradation demonstrated by 40 mg/mloligomer matrices resulted in a diffusion only 10 kDa release mechanism.In contrast, all matrix formulations tested exhibited 2 MDa FITC-dextranrelease mechanisms governed by both diffusion and degradation.

Evaluation of density dependent release of 10 kDa FITC-dextran inabsence of collagenase also showed a progressive increase in T50% valuesas a function with oligomer concentration or matrix density as shown inFIG. 18 . As expected T50% values obtained in absence of collagenasewere less than those measured in the presence of collagenase (FIG. 17 ).Weibull parameters indicated that 10 kDa FITC-dextran release involvedprimarily diffusion for 3 mg/ml and 20 mg/ml, while release from 40mg/ml matrices involved diffusion and some other mechanism. Thisexperiment further validated and defined how densifying collagenmatrices modulate molecular release from collagen-fibril matrices.

Example 12: Preparation and Characterization of Collagen Polymers

Market weight porcine hides were obtained from commercialmeat-processing sources according to Purdue University Animal Care andUse Committee (PACUC) guidelines. Oligomeric collagen was extracted fromthe dermis as described previously (Kreger et al, (2010) Biopolymers93:690-707, herein incorporated by reference). Monomer-rich(telocollagen) collagen was prepared by extracting pig skin with 0.5 Macetic acid followed by salt precipitation. Telopeptide regions withinthe collagen molecule, which contain intermolecular cross-linking sites,were enzymatically removed by complete pepsin digestion. All collagenswere dialyzed exhaustively against 0.1 M acetic acid and thenlyophilized. Prior to use, lyophilized collagens were dissolved in 0.01N HCl. All collagens were rendered aseptic by exposure to chloroformovernight at 4° C. Collagen concentration was determined using a SiriusRed (Direct Red 80) assay. The collagen formulations were standardizedbased upon purity as well as polymerization capacity. Here,polymerization capacity is defined as the relationship between the shearstorage modulus (G′) of the polymerized matrices and the collagencontent of the polymerization reaction. Commercial monomer collagen,acid solubilized type I collagen harvested from rat tails was purchasedfrom BD Biosciences (Bedford, Mass., referenced as BD-rat tail collagen,BD-RTC).

Example 13: Preparation of Collagen-Fibril Matrices

For preparation of 3D collagen fibrillar matrices, collagens werediluted in 0.01N HCl and further neutralized with phosphate bufferedsaline (PBS, 10×, pH 7.4) and 0.1N sodium hydroxide (NaOH) to achieveneutral pH (7.4) and a final collagen concentration of 3 mg/ml. Forformulating matrices with FITC-Dextran, a similar polymerization processwas applied and FITC-Dextran (10 kDa, 40 kDa, 500 kDa, or 2 MDaInvitrogen, Eugene, Oreg.) was predissolved in 10× PBS to yield a finalconcentration of 0.5 mg/ml within the polymerized matrix. Preparation ofmatrices with varied oligomer:monomer ratios involved neutralization ofeach component at 3 mg/ml with 0.5 mg/ml FITC-Dextran and then varyingthe volume ratio to achieve 0:100, 25:75, 50:50, 75:25, and 100:0 priorto polymerization.

The neutralized collagen solutions were kept on ice prior to theinduction of polymerization by warming to 37° C. In other experimentsthe temperature of the rheometer plate was maintained below 10° C. for aperiod of 5 minutes to get the baseline storage modulus ofnon-polymerized matrices and then increased to 37° C. Due to theincreased viscosity of the collagen solutions, positive displacementpipettes (Microman, Gilson, Middleton, Wis.) were used to accuratelypipet all collagen solutions. To confirm that the addition ofFITC-Dextran had no effect on collagen polymerization kinetics andmatrix physical properties, matrices were prepared in the absence andpresence of FITC-Dextrans (10 KDa and 2 MDa, 0.5 mg/ml). Time-dependentchanges in viscoelastic properties (shear storage modulus (G′), shearloss modulus (G″), and phase shift delta (/5) during polymerization weremeasured in oscillatory shear using an AR2000 rheometer (TA Instruments,New Castle, Del.) adapted with a stainless steel 40 mm diameter parallelplate geometry). Polymerization kinetics and viscoelastic properties formatrices prepared in the presence and absence of FITC-dextran then werecompared.

Example 14: Measurement of Release Kinetics

Collagen matrices (3 mg/ml) containing various sized FITC-Dextran at aconcentration of 0.5 mg/ml were polymerized in 48-well plates (250J.lllwell). Samples then were overlaid with 750 μl PBS, pH 7.4containing no collagenase or 125 U/ml bacteria Clostridium histolyticumcollagenase (CLS4, Worthington Biomchemical Corporation, Lakewood,N.J.). Plates were subjected to gradual rotation at 60 rpm on a FisherScientific Clinical Rotator. The supernatant from each sample wascollected at specific time intervals and replaced with fresh solutions.Sampling intervals were predicted based on simulated release curves foreach molecule size, modeled using diffusion equation given by Siepmannet al. A spectrofluorometer (Molecular Devices Spectramax M5) was usedto measure fluorescence at an excitation and emission wavelength of 493and 530 nm respectively. This process was repeated until RelativeFluorescence Units from supernatant of wells matched baselinefluorescence (PBS plus/minus collagenase containing no FITC-Dextran),indicating completion of the FITC-Dextran release.

The data collected were used to plot % Cumulative release vs time inminutes. Two parameters were used to define release curves-rate ofrelease and T50% of Release. Initial rate of release was defined as theslope of the release curve analyzed over time required for reaching 25%of cumulative release, obtained using linear trendline fit in MicrosoftExcel. T50% of Release, defined as time required to obtain 50% ofCumulative Release, was obtained from power best fits of release curvesin Matlab (Mathworks).

All statistical analyses were performed in MiniTab. The comparisonbetween collagenase and PBS was performed with a 2-sample Student'sT-Test with a confidence interval of 95%. The comparisons between drugsize and matrix composition were performed with ANOVA and post-hoc Tukeytest with a 95% confidence interval.

While the inventions have been illustrated and described in detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly certain embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected.

1. A collagen-based therapeutic delivery device comprising: an insolublesynthetic collagen-fibril matrix comprising a polymerization product ofa mixture of soluble oligomeric collagen with one or more type ofnon-oligomeric soluble collagen molecules; and a first active agentdispersed throughout the collagen-fibril matrix or within a portion ofthe collagen-fibril matrix; wherein the collagen-fibril matrix exhibitsa stiffness of at least 5 Pa.
 2. The therapeutic delivery device ofclaim 1, wherein the collagen-fibril matrix comprises type I collagen.3. The therapeutic delivery device of claim 1, wherein the one or moreother type of non-oligomeric soluble collagen molecules comprises one orboth of soluble telocollagen molecules and soluble atelocollagenmolecules.
 4. The therapeutic delivery device of claim 1, wherein thecollagen-based therapeutic delivery device is a tissue graft.
 5. Thetherapeutic delivery device of claim 1, wherein the collagen-basedtherapeutic delivery device is lyophilized.
 6. The therapeutic deliverydevice of claim 1, wherein the collagen-fibril matrix comprises apolymerization product of a mixture of soluble oligomeric collagen withone or more type of non-oligomeric soluble collagen molecules andwherein the oligomeric collagen and total non-oligomeric solublecollagen molecules are in a ratio within a range selected from the groupconsisting of 5:95 to 10:90, 10:90 to 15:85, 15:85 to 20:80, 20:80 to25:75, 25:75 to 50:50, and 50:50 to 75:25.
 7. The therapeutic deliverydevice of claim 1, further comprising a second active agent dispersedthroughout the collagen-fibril matrix or within a portion of thecollagen-fibril matrix.
 8. The therapeutic delivery device of claim 7,wherein each of the first and second active agents is a growth factor ora drug.
 9. The therapeutic delivery device of claim 1, wherein thecollagen-fibril matrix includes a first portion having a first densityand a second portion having a second density; wherein the first densityis different than the second density.
 10. The therapeutic deliverydevice of claim 1, wherein the collagen-fibril matrix includes a firstportion having dispersed therein the first active agent and a secondportion having dispersed therein a second active agent, wherein thetherapeutic delivery device exhibits a first release profile for thefirst active agent and a second release profile for the second activeagent, and wherein the first release profile is different than thesecond release profile.
 11. A method for making a therapeutic deliverydevice, comprising: forming an aqueous solution of building blockscomprising a first quantity of soluble collagen-fibrils and solublenon-oligomeric collagen molecules; causing the building blocks topolymerize by self-assembly, thereby forming an insoluble polymerizationproduct comprising a mixture of oligomeric collagen and non-oligomericsoluble collagen molecules; and either including a second quantity of anactive agent in the aqueous solution whereby said causing step forms theinsoluble synthetic collagen-fibril matrix having the active agentdispersed therein or contacting the insoluble synthetic collagen-fibrilmatrix with the second quantity of the active agent to form acollagen-fibril matrix having the active agent dispersed therein;wherein the collagen-fibril matrix exhibits a stiffness of at least 5Pa.
 12. (canceled)
 13. The method of claim 11, further comprisingcompressing the insoluble synthetic collagen-fibril matrix to form acondensed insoluble synthetic collagen-fibril matrix.
 14. The method ofclaim 13, wherein said compressing comprises subjecting the insolublesynthetic collagen-fibril matrix to confined compression.
 15. The methodof claim 11, further comprising, after said causing step, lyophilizingthe insoluble synthetic collagen-fibril matrix.
 16. A pre-matrixcomposition comprising an aqueous solution of building blocks includinga first quantity of soluble collagen-fibrils and soluble non-oligomericcollagen molecules; and a second quantity of an active agent in theaqueous solution, wherein wherein the building blocks are operable toself-assemble into a macromolecular insoluble synthetic collagen-fibrilmatrix having a stiffness of at least 5 Pa in the absence of anexogenous cross-linking agent.
 17. The pre-matrix composition of claim16, wherein the one or more other type of non-oligomeric solublecollagen molecules comprises one or both of soluble telocollagenmolecules and soluble atelocollagen molecules.
 18. The pre-matrixcomposition of claim 16, wherein the oligomeric collagen comprises typeI collagen.
 19. The pre-matrix composition of claim 16, wherein theactive agent is a growth factor or a drug.
 20. The pre-matrixcomposition of claim 16, wherein the active agent is covalently attachedto one or more of the building blocks.
 21. The pre-matrix composition ofclaim 16, wherein the pre-matrix composition comprises the oligomericcollagen and total non-oligomeric soluble collagen molecules in a ratiowithin a range selected from the group consisting of 5:95 to 10:90,10:90 to 15:85, 15:85 to 20:80, 20:80 to 25:75, 25:75 to 50:50, and50:50 to 75:25. 22-25. (canceled)